Mechanism of phytochrome action in the control of biosynthesis of anthocyanin in Brassica oleracea

-The synthesis of anthocyanin in red-cabbage is very sensitive to control by light, R/FR reversibility being effected by exposures of 5 min duration, The demonstration of this control does not depend upon a preceding irradiation of high-intensity light but depends upon the duration of incubation in darkness subsequent to irradiation. R/FR reversibility is well shown in seedlings kept in darkness for 48 hr after exposure but after 120 hr this reversibility is no longer evident. This is due to the fact that a further synthesis of anthocyanin occurs in unexposed seedlings and in FR and R/FR treated material in the period from 48 to 120 hr but does not occur in the R treatment after 48 hr. Reagents such as n-propanol which are believed to increase membrane permeability, greatly increase anthocyanin synthesis in dark grown material. n-PrOH also reverses the effect of 5 min FR irradiation but, by contrast with R light, does not promote PAL activity. It is concluded that the limitation to synthesis in material unexposed to light is substrate availability at the site of flavonoid biosynthesis, rather than the level of PAL activity. The evidence presented supports the hypothesis that R/FR reversible phytochrome action involves the control of the passage of substrate through a membrane to the site of anthocyanin biosynthesis.     

The effect of kinetin in relation to photocontrol of anthocyani biosynthesis in Brassica oleracea

Kinetin stimulates the synthesis of anthocyanin in dark-grown seedlings of red-cabbage. When applied for only 15 min its effect resembles that of 5 min R light and can be nullified by a subsequent exposure to 5 min FR. However, kinetin fails to stimulate PAL activity in the dark-grown seedlings. It is suggested that the effect of kinetin, like that of R light, may be to increase membrane permeability, allowing a pool of endogenous substrate to reach the site of anthocyanin biosynthesis.     

Membrane permeability changes: A component of phytochrome-mediated anthocyanin synthesis in Sinapis alba

Dark synthesis of anthocyanin in Sinapis alba seedlings is greatly promoted by short treatments with n-propanol. The effect of n-propanol treatment is reversed by subsequent far-red light pulses. Photoreversibility kinetics suggest that n-propanol and red light act in the same way. However, phytochrome measurements and other control experiments suggest that n-propanol treatment does not lead to significant Pfr production, nor does it increase the effectiveness of any ‘dark’ Pfr present in the seedlings. The findings are difficult to explain in terms of Pfr as the sole effector of this phytochrome-mediated response.     

Change in sensitivity to far-red irradiation on anthocyanin biosynthesis in red cabbage seedlings

Abstract. Anthocyanin biosynthesis in red cabbage seedlings has been shown to be under a red/far-red (R/FR) reversible phytochrome control. The 'escape' of anthocyanin synthesis from FR photoreversibility occurred between 12 and 20 h. However, a similarity exists between the escape curve and the temporal change in sensitivity to FR alone, which suggests that some of the 'escape' may be due to the promotive effect of FR. Some escape times may not therefore be a true measure of the duration of Pfr action, contrary to the traditional view.     

Investigation of Brassica oleracea and Nasturtium officinale seeds for the presence of epithiospecifier protein

Cabbage seeds contain 5 glucosinolates and on autolysis produce, in particular, 1-cyano-2-3-epithiopropane. Watercress seeds contain 4 glucosinolates, but none capable of forming a cyanoepithioalkane. Cabbage exhibited behaviour commensurate with possession of an epithiospecifier protein (ESP) whilst watercress did not show any such activity.     

Pectic polysaccharides of cabbage (Brassica oleracea)

Pectic substances extracted from cabbage cell walls with water, at 80°, and (NH₄)₂C₂O₄, at 80°, accounted for 45%(w/w) of the purified cell wall material. Only a small amount of neutral arabinan was isolated. Partial acid hydrolysis and methylation analysis revealed that the major pectic polysaccharide had a rhamnogalacturonan backbone to which a highly branched arabinan was linked, at C-4 of the rhamnose units, mainly through short chains of (1→4)-linked galactopyranose residues. The bulk of the soluble pectic substances had only small amounts of proteins associated with them. After further extraction of the depectinated material with 1M and 4M KOH, to remove the hemicelluloses, the cellulose residue was found to contain a pectic polysaccharide which was solubilized by treatment with cellulase. The general structural features of the pectic polymers are discussed in the light of these results.     

Effect of temperature on phytochrome—mediated anthocyanin synthesis in red cabbage

Abstract. A brief high temperature treatment (45 C) promoted anthocyanin synthesis in 2-d-old dark-grown red cabbage seedlings. The increased effectiveness of a temperature/red light treatment as opposed to the reverse sequence suggests that an elevated temperature 'induces' some component which facilitates the phytochrome response.     

Structural investigation of an arabinan from cabbage (Brassica oleracea var. capitata)

An arabinan has been isolated from the hot water-soluble pectic substances of cabbage cell wall material. Methylation analysis involving GC-MS of methylated alditol acetates formed from the methylated arabinan has shown that the parent polysaccharide is highly branched and of the same structural type as other arabinans associated with seed pectins.     

Wax synthesis in Brassica oleracea as modified by trichloroacetic acid and glossy mutations

Two different mutations in Brassica oleracea, gl₅ and gl₄ have been re-investigated using acetate-1-¹⁴C labelling in an attempt to define more closely the nature of the genetic blocks to wax synthesis. It has been found that gl₅ is a mutation which blocks elongation in the Step C₂₈–C₃₀. The mutation gl₄ exhibits no elongation block and could be blocked in the decarboxylation Step C₃₀–C₂₉. 0·1 mM TCA supplied in the culture solution of cauliflower seedlings affected the leaf surface by producing a glossy appearance similar to that induced by gl₃ and gl₄. At this concentration growth was not inhibited and the appearance of the plants was normal except for the surface wax. The amount of surface wax produced was about 40% of that in untreated seedlings on a leaf area basis. Slight, but significant changes in wax composition were noted, mainly involving a reduction in C₃₀ acids and aldehydes, a slight reduction (33–29%) in alkane content, and a marked difference in chain length composition of the alkanes with C₂₇ increased relative to C₂₉. Over a range of concentrations from 0·1–1 mM, TCA inhibited incorporation of label from acetate-1-¹⁴C into C₃₀ acids and aldehydes more than into C₂₈ at concentrations 0·4–0·8 mM while label tended to accumulate in C₂₄ and C₂₆ acids; thus elongation C₂₈–C₃₀ was especially sensitive to TCA. TCA also inhibited incorporation into primary alcohols and esters almost as much as into C₂₉ compounds. In spite of relatively specific effects on incorporation of label into longer chain lengths, the resulting block to C₃₀ synthesis is not sufficient to make much difference to the overall rate of C₂₉ synthesis. Both results of analysis of wax from whole plants and experiments with tissue slices in vitro indicated that the effect of TCA in reducing the glaucousness of the leaf surface is a combination of overall reduction of wax synthesis together with slight but significant changes in wax composition.     

Light induced changes in anthocyanin concentration,activity of phenylalanine ammonia-lyase and flavanone synthase and some of their properties in Brassica oleracea

Seedlings of red cabbage, Brassica oleracea cv Red Danish, germinated in the dark, rapidly produced anthocyanins upon illumination. The anthocyanin production increased up to six days of illumination time. The activity of phenylalanine ammonia-lyase increased rapidly in illuminated seedlings to a maximum at 8 hr and declined thereafter to dark levels. During this period the activity of flavanone synthase, the first enzyme responsible for the establishment of C₁₅ flavonoid skeleton, paralleled that of the anthocyanin concentration. The crude flavanone synthase has a pH optimum at around 8, a molecular weight of ca 120 000, and is able to utilize only p-coumaryl-CoA as co-substrate for the production of flavonoids.     

A chemotaxonomic study of Brassica oleracea with particular reference to its relationship to Brassica alboglabra

A chemotaxonomic study has been made of Brassica oleracea L., its horticultural varieties and Brassica alboglabra Bailey with reference to seed proteins, certain isoenzymes and isothcioyanates. The results of the investigation do not support separate species status in respect of B. alboglabra.     

Occurrence,location and development of anthocyanoplasts

Intensely pigmented organelles (anthocyanoplasts) have been found in anthocyanin-producing cells of more than 70 species representing at least 33 families of angiosperms. When fully developed these structures are typically spherical and normally only one is present in each pigmenented cell. The development of the anthocyanoplast has been studied in both light and dark-grown red cabbage seedlings and the location of the mature organelle has been shown, by the use of isolated protoplasts and vacuoles, to be within the main cell vacuole. Evidence is presented which suggests that the anthocyanoplast is membrane-bounded and that it is the site of anthocyanin biosynthesis.     

Phosphatases from pollen of Brassica campestris and Lilium regale

Soluble and wall-bound acid phosphatases isolated from rape seed pollen showed similar properties except for the pH optimum curve which was elevated for the cell wall enzyme. About 50 % of the phosphatase activity of washed pollen wall preparations could be solubilized with Triton X-100, compared with only ca 20% for the corresponding preparation from lily pollen. A comparison of the wall-bound acid phosphatase of rape seed and lily pollen showed a marked difference in specificity towards fructose-6-phosphate and glucose-6-phosphate. A Mg²⁺-dependent alkaline pyrophosphatase was obtained from rape seed pollen but this activity could not be detected in cell wall preparations.     

Ozone fumigation results in accelerated growth and persistent changes in the antioxidant system of Brassica oleracea L. var. capitata f. alba

The growth response and antioxidant capacity of Brassica oleracea var. capitata f. alba plants treated with 70 ppb of ozone was examined. Four week old cabbage seedlings were fumigated with O₃ for 3 days before being transplanted into the growing field. The effect of O₃ treatment was determined directly after fumigation and over the course of field cultivation. Plants subjected to O₃ treatment had an increased diameter of rosettes and number of leaves after 3 and 7 weeks in agriculture, respectively. In addition, the vast majority of fumigated plants reached marketable quality faster than control plants, indicating a positive role of episodes of increased O₃ concentrations during vegetation on growth and yielding.     

Anthocyanins in Salix species: A new anthocyanin in salix purpurea bark

Cyanidin 3-glucoside and delphinidin 3-glucoside (minor component) occurred in the bark of Salix purpurea (29 cultivars examined), S. fragilis (3 cvs), S. americana (4 cvs), S. rubra (1 cv.), S. incana (1 cv.), S. aegyptica (1 cv.) and S. alba (3 cvs) and several hybrids. A previously unrecorded anthocyanin was also present in appreciable amounts in the bark of eleven S. purpurea cultivars, notably those with small leaves. In other large-leaved cultivars of S. purpurea (18) it occurred only in traces or was not detected with certainty. A trace was found in one purpurea hybrid, but it was not detected in four others. Small amounts of the new anthocyanin were also found in S. incana (1 cv.), S. fragilis cv. Basfordiana, S. americana cv. Cordata and one hybrid, but were absent from other cultivars of S. fragilis (2) and S. americana (2), and also from S. alba (3), S. aegyptica (1), S. daphnoides (1), S. rubra (1), S. triandra (1), S. viminalis (1) and three hybrids. The new anthocyanin is unique in containing fructose as well as glucose and is based upon a previously undescribed anthocyanidin, possibly dimeric in nature, which is provisionally named purpurinidin.     

Phytochrome control of amino acid synthesis in cotyledons of Sinapis alba

Continuous far red light, acting through phytochrome, stimulates the rate of incorporation of density label into amino acids in the cotyledons of Sinapis alba. It is shown that such stimulation leads to increased incorporation of label into proteins. This has important consequences for experiments in which rates of enzyme synthesis in light treated and dark grown plants are compared by labelling methods. The results of some such experiments are re-evaluated.     

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Effect of ethylene and metabolic inhibitors on anthocyanin biosynthesis

The use of metabolic inhibitors indicated that ethylene-enhancement of light-induced anthocyanin biosynthesis in Sorghum vulgare is through promotion of enzyme synthesis. Ethylene treatment had no effect on the amount of cyanidin synthesized in sorghum tissue infiltrated with actinomycin D to inhibit RNA synthesis. Treatment of sorghum tissue with ethylene in the dark for 24 hr prior to light-induction of anthocyanin biosynthesis reduced the ability of cycloheximide to inhibit anthocyanin formation in the tissue. Ethylene treatment promoted the biosynthesis of two 3-deoxyanthocyanidins in sorghum for which light-induced RNA synthesis is not necessary.     

Malonic acid: The acyl moiety of the Mimulus luteus anthocyanin

Malonic acid has been identified as the acylating agent of the anthocyanin produced by the Mimulus luteus complex.     

Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures, was not observed with the H. gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation.